Deparaffinization and rehydration of 5 μm FFPE meningioma xenograft sections and hematoxylin and eosin staining were performed using standard procedures. Immunostaining was also performed on 5 μm FFPE meningioma xenograft sections using an automated Ventana Discovery Ultra staining system. Immunohistochemistry was performed using rabbit monoclonal Ki-67 (Ventana, clone 30-9, Cat# 790-4286, 1:6) with incubation for 16 min. Histologic and immunohistochemical features were evaluated using light microscopy on an BX43 microscope with standard objectives (Olympus). Images were obtained and analyzed using the Olympus cellSens Standard Imaging Software package.
Clone 30 9
Manufactured by Roche
Sourced in United States
The Clone 30-9 is a laboratory equipment product designed for general research applications. It serves as a tool for various tasks in life science and biomedical research. The core function of the Clone 30-9 is to provide a reliable and consistent platform for performing specific laboratory procedures. Further details on the intended use or capabilities of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Cellsens standard imaging software package, by Olympus (1 mentions)
Bx43 microscope, by Olympus (1 mentions)
Benchmark xt, by Roche (1 mentions)
Nanozoomer 2.0 ht scanner, by Hamamatsu Photonics (1 mentions)
Clone 4b5, by Roche (1 mentions)
Clone sp1, by Roche (1 mentions)
Iscan coreo, by Roche (1 mentions)
6 protocols using clone 30 9
1
Immunohistochemical Analysis of Meningioma Xenografts
2
Immunohistochemical Assessment of Ki-67 in Lung SCC
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Immunohistochemical staining was performed using a Ventana Benchmark XT automated multifunctional slide system (Ventana, Arizona, USA), and a monoclonal antibody against Ki-67 (clone 30-9, Ventana Medical Systems, Inc., USA) was used. The number of tumor cells per sample was calculated by counting and scoring the percentage of positive cells in 5 randomly selected fields at high magnification (400×). The number of Ki-67-positive tumor cells per 100 tumor cells was counted in each microscopic field. The percentage of positive tumor cells was scored to assess Ki-67 immunostaining as follows: positive if brownish-yellow staining was seen in the nucleus and Ki-67-positive cells were > 5%; negative if no brownish-yellow staining was observed or Ki-67-positive cells were < 5%. Next, Ki-67 expression was scored according to the percentage of positive cells: 1 for < 15%, 2 for 16% - 45%, 3 for 46% - 75%, and 4 for > 75% (Figure 1 ).![]()
Abbreviation : SCC, squamous cell carcinoma.
Immunohistochemical staining of Ki-67 expression in advanced lung SCC. (magnification 400×). (
3
Ki-67 Immunohistochemistry Staining Protocol
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Commercially available antibody raised against Ki-67 (clone 30-9, Ventana Medical Systems Inc., Tucson, AZ, USA, 1:200) was used on a Ventana Benchmark automated staining system. For Ki-67, only nuclear staining was regarded to be specific. Immunohistochemistry was evaluated, if possible, in two cores per tumor and the results were combined. Tissue cylinders with a diameter of 0.6 mm and a height of 3–4 mm, corresponding to approximately 2800 pixels, were punched from representative tumor areas of a “donor” tissue block using a custom-built instrument. The Images were digitized using a Hamamatsu NanoZoomer 2.0-HT scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) and stored as jpegs with 0.235 pixels/micrometer using NDI Viewer (NewTek Inc., San Antonio, TX, USA).
4
TNBC Immunohistochemistry and TILs Assessment
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Immunohistochemical analysis on BC specimens were performed at our Pathology Department and tumors were defined as TNBC if ER (Ventana, Clone SP1, pre-diluted) and PgR (clone 30-9; Ventana Medical Systems Inc, pre-diluted) IHC staining was ≤ 1%, together with 0/1 + score HER2 on IHC (clone 4B5; Ventana Medical Systems Inc, pre-diluted) and/or non-amplified on fluorescent in situ hybridization (FISH) for 2 + score HER2 cases. For each study patient, formalin-fixed paraffin-embedded (FFPE) tumor samples were retrieved from Institutional Pathology archives. Stromal TILs were assessed according to consensus guidelines7 (link),30 (link) by one investigator (BF) who was blinded to clinical data. AR expression was measured by IHC, using an anti-androgen receptor (Clone SP107; Ventana Medical Systems Inc, pre-diluted) and the percentage of AR-positive nuclei was quantified. A cutoff of 10% or greater was used to define an AR-positive tumor31 (link). The IHC threshold (= 0 + or < 10% or ≥ 1 + and ≥ 10%) was used for AR.
5
Immunohistochemical Evaluation of Breast Cancer Markers
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Needle biopsies were processed following current ASCO/CAP guidelines for optimal tissue handling. Three-micrometer-thick sections were obtained from the blocks and subjected to heat-induced antigen retrieval. Immunochemical staining of Ki67 was performed using a prediluted rabbit monoclonal antibody against human Ki67 (Clone 30-9, Ventana, Tucson, AZ, USA) and carried out in a Benchmark Ultra stainer using an UltraView detection kit. A similar procedure was also used for estrogen (clone SP1) and progesterone receptors (clone 1E2) and HER2 (clone 4B5).
6
Immunohistochemical Analysis of Cortical Pathology
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The Ki67 immunohistochemistry was performed on a benchmark ULTRA using clone 30-9 (Ventana Medical Systems, Tucson, AZ), and interpreted with an iScan Coreo and Virtuoso algorithm (Ventana Medical Systems). A histopathological analysis of the tissue samples was performed to validate their use in the rest of the study. The architectural disorganisation of the cortex and neuronal heterotopia were studied using NeuN immunostaining. Microglia activation was examined by Iba-1 marking. The experiments were performed using the protocols described later.
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